Lysergic acid diethylamide (LSD) is a potent classic serotonergic psychedelic, which facilitates a variety of altered states of consciousness. Here we present the first meta-analysis establishing dose-response relationship estimates of the altered states of consciousness induced by LSD. Data extracted from articles identified by a systematic literature review following PRISMA guidelines were obtained from the Altered States Database. The psychometric data comprised ratings of subjective effects from standardized and validated questionnaires: the Altered States of Consciousness Rating Scale  and the Mystical Experience Questionnaire (MEQ30). We performed meta-regression analyses using restricted cubic splines for data from studies with LSD doses of up to 200 μg base. Most scales revealed a sigmoid-like increase of effects, with a plateauing at around 100 μg. The most strongly modulated factors referred to changes in perception and illusory imagination, followed by positively experienced ego-dissolution, while only small effects were found for Anxiety and Dread of Ego Dissolution. The considerable variability observed in most factors and scales points to the role of non-pharmacological factors in shaping subjective experiences. The established dose-response relationships may be used as general references for future experimental and clinical research on LSD to compare observed with expected subjective effects and to elucidate phenomenological differences between psychedelics. The terN,N-DMT) –6. On thN,N-DMT) , and LSD targets .After its initial synthesis in 1938, LSD’s psychedelic properties have accidentally been discovered in 1943 by the Swiss pharmacologist Albert Hofmann . Most inLSD has recently been re-evaluated for the treatment of different mental health conditions, like anxiety and depression in patients with , 16 and set and setting effects [The data to establish dose-response relationships for LSD is limited by the availability of studies that cohere to current research standards, as LSD has only recently returned to basic and clinical research. The range of LSD doses employed in current research is on average markedly below the doses administered in studies from the 1950 to 70 s. At that time, high doses mostly ranging between 400 and 600 μg were applied, especially in the treatment of alcohol use disorder, whereas in recent studies doses of 200 μg have not been exceeded, neither in healthy subjects , nor in  effects , 30. Crohttp://alteredstatesdb.org, [With the present meta-analysis, we aim to obtain estimates of the relationship between LSD doses and the quality of psychedelic experiences. The data stem from the Altered States Database , which sdb.org, . Availabhttps://osf.io/8mbru, version ASDB_v.2022-12-31), which contains data from MEDLINE-listed studies on altered states of consciousness published from 1975-01-01 to 2022-12-31. The ASDB stems from a systematic literature review according to PRISMA standards [Psychometric questionnaire data on the subjective experience of LSD were included in this meta-analysis. The data has been retrieved from the ASDB repository on Open Science Framework  Dread of Ego Dissolution, (3) Oceanic Boundlessness, (4) Visionary Restructuralization, and (5) Vigilance Reduction. In the more recent 11-ASC version  Spiritual Experience, (3) Blissful State, (4) Insightfulness, (5) Disembodiment, (6) Impaired Control and Cognition, (7) Anxiety, (8) Complex Imagery, (9) Elementary Imagery, (10) Audio-Visual Synesthesia, and (11) Changed Meaning of Percepts. Both analysis schemes have been validated and demonstrate good reliability  Positive Mood, (3) Transcendence of Time and Space, and (4) Ineffability. This factor structure is currently recommended for analyses and has been assessed for reliability, yielding good scores for all four subscales  [This meta-analysis included psychometric data from commonly applied questionnaires to assess the phenomenology of altered states of consciousness, namely from two versions of the Altered States of Consciousness Rating Scale (the 5D-ASC and the 11-ASC), and from the Mystical Experience Questionnaire (MEQ30). The Altered States of Consciousness Rating Scale –49 is a g Scale” ), only 4.88–0.95 , 53); 11.88–0.95 ). The My.88–0.95 , consist80–0.95) , 56. A m80–0.95) , Majić e80–0.95) .p = 0.8 for the variance-covariance matrix. The analyses were performed in R version 4.2.2 [https://github.com/TimHirschfeld/doseresponse_LSD.We performed meta-regression analyses using restricted cubic splines. A restricted cubic spline model is non-linear and consists of a series of piecewise cubic polynomials that are connected at the position of the knots, with a linear curve before the first and after the last knot . It was on 4.2.2  with theon 4.2.2  using thon 4.2.2 . Clusteron 4.2.2  in the RSixteen studies assessing the effects of LSD were included in this analysis, with overall 765 LSD applications. Table https://dose-response-lsd.asdb.info/.Radar charts for each questionnaire and dose-response relationships for each factor and scale of the respective questionnaires are presented in Fig.  as well as positively experienced dissolution of boundaries between self and surroundings (Oceanic Boundlessness), whereas Anxiety, Auditory Alterations and Dread of Ego Dissolution were barely modulated. On the MEQ30, the strongest dose-modulation was found for Ineffability and the weakest for Mystical.A meta-analysis on psychometric data was performed to estimate dose-response relationships of subjective effects of LSD. The analysis included data from applications of doses from 4 to 200 μg LSD base, in healthy and clinical study participants. Given that the data is based on retrospective subjective reports relying on introspection, we did not employ assumptions on the specific shape of the fitted model ; instead, we opted for fitting restricted cubic splines that allow for flexible model fits. Most of the fitted dose-response functions closely resemble sigmoid curves, which is expected and plausible for biophysiological processes. We established robust dose-response relationships, in which LSD dose increased effects on all scales and factors of the included questionnaires. On the 5D- and 11-ASC, the strongest dose-modulation was found for items referring to changes in perception and imagination Auditory Alterations, Dread of Ego-Dissolution, Anxiety) increase rather linearly. This is in line with a dose-response, within-subject study by Holze et al. (2021) that found similar rating scores for 100 µg and 200 µg for Blissful State, Insightfulness, and Changed Meaning of Percepts [Dread of Ego Dissolution (reflecting disembodiment and fear) [Most factors and scales exhibit a sigmoid-like shape of dose-effects that either flattens out or reaches a plateau at around 100 μg, while scales and factors with relatively small dose-modulation  , indicatnd fear) . Despitend fear) . AccordiAudio-Visual Synesthesia, Complex and Elementary Imagery) show an x-axis intercept at around 10 μg, and at 20 μg reach around 10% of the maximum score. These effects are unlikely due to placebo effects, as those of the given studies which included a placebo condition reported only negligible effects [LSD doses below 10 μg  or 20 μg effects , 66–69. A recent systematic review indicates a mediating role of mystical-type experiences for treatment outcomes of psychedelic therapy , so specInsightfulness, Spiritual Experience, Blissful State, and Complex Imaginary) [Non-pharmacological factors that influence psychedelic experiences are often categorized in set  and setting (environment of substance administration) , 72 and aginary)  comparedaginary) , 22, 28.Further limitations should be considered when using the analysis at hand to determine LSD doses. First, administration methods varied across studies. Most studies administered LSD orally as capsules, while it was administered sublingually in two studies , 45 and In conclusion, the present meta-analysis on subjective effects of LSD in study participants revealed dose-dependent alterations of consciousness with substantial heterogeneity of effects across laboratory and therapeutic settings on all scales and factors assessed by given questionnaires. The strongest dose-modulation was found for perceptual and imaginative changes followed by positively experienced dissolution of boundaries between self and surroundings with a limited spiritual or mystical-type character in the given dosage range. Dose-response relationships resemble sigmoid functions on most factors and scales of the questionnaires, with an observable ceiling effect at circa 100 μg LSD base. Results may be used as a general reference to relate observed with expected dose-specific effects.Supplement PRISMA Flowchart


Furthermore, nanoSIMS measurements revealed substantial incorporation of polyethylene-derived carbon into fungal biomass. Our results demonstrate the potential of R. mucilaginosa to mineralize and assimilate carbon from plastics and suggest that fungal plastic degradation may be an important sink for polyethylene litter in the marine environment.Ocean plastic pollution is a severe environmental problem but most of the plastic that has been released to the ocean since the 1950s is unaccounted for. Although fungal degradation of marine plastics has been suggested as a potential sink mechanism, unambiguous proof of plastic degradation by marine fungi, or other microbes, is scarce. Here we applied stable isotope tracing assays with  Ideonella sakaiensis hydrolyzes polyethylene terephthalate [Rhodococcus ruber strain C208 has been found to degrade polyethylene (PE) and polystyrene (PS) [Bacillus sphericus and Bacillus cereus have been shown to degrade polyethylene [Aspergillus and Penicillium species, isolated from soils and gut microbiomes, were shown to degrade polyethylene [Zalerion maritimum [Alternaria alternata [Ocean plastic pollution is an environmental problem of exponentially increasing magnitude –6. Howevhthalate –28, whilene (PS) –31. In aethylene . In contethylene , oil, anethylene , 35—i.e.ethylene –38, whicethylene , 40. Witethylene –46. Thouethylene –51, onlyaritimum  and Altelternata , have beA general problem in measuring microbial plastic degradation and comparing results from different studies stems from methodological challenges and limitations. The chemical configuration of the used polymers, for example, the degree of crosslinking, crystallinity, and the addition of additives, as well as the size of the plastic particles and environmental/incubation conditions, likely affect microbial degradation . The mosRhodotorula mucilaginosa, which we isolated from plastic debris from a North Sea laboratory microcosm, is capable of degrading UV-treated polyethylene. We incubated this fungus with virgin (-UV) and UV-irradiated 13C-labeled polyethylene and traced the polyethylene-derived carbon from the polymer source to the terminal oxidation product CO2 and measured polyethylene mineralization rates with unprecedented sensitivity. Furthermore, we visualized the assimilation of plastic-derived 13C into single cells of R. mucilaginosa by nanometer scale secondary ion mass spectrometry (nanoSIMS). This approach allows following the fate of plastic-derived carbon in marine ecosystems.Here, we show that the fungus Rhodotorula mucilaginosa , either UV-treated or untreated, and measuring the transfer of 13C-label into the CO2 pool and cellular biomass (<1 week of incubation). The first assay was performed using 13C-labeled polyethylene as the sole energy and organic carbon source. In this experiment, the cultured cells were washed twice with sterile and autoclaved seawater, centrifuged (4000×g) at room temperature for 5 min, and “starved” for a week in sterile seawater at 25 °C to allow consumption of potentially remaining sucrose from medium prior to the incubation with polyethylene. In the second assay, we tested the ability of R. mucilaginosa to utilize polyethylene in the presence of another, more labile carbon source. In this setup, in addition to 13C-labeled polyethylene, the incubations also contained sucrose-based culture medium . Both assays included three treatments, each performed in triplicates: (i) 13C-labeled polyethylene and R. mucilaginosa, (ii) UV-treated 13C-labeled polyethylene and R. mucilaginosa, and (iii) uninoculated control with UV-treated 13C-labeled polyethylene.Details of the experimental setups are provided in the Supplementary appendix. In brief, 13C-CO2 in the incubation bottles over time. Firstly, the identity and concentration of the headspace CO2 gas were measured using gas chromatography with quadrupole mass spectrometry and flame ionization detection, respectively. The isotopic composition of headspace CO2 was then determined by isotope ratio mass spectrometry . The pH of the liquid phase was determined with a pH meter (Mettler-Toledo Seven compact S210) at the end of the incubation. Concentrations of dissolved inorganic carbon (DIC) in the liquid phase were then determined from headspace CO2 and pH measurements [2 and DIC concentrations were then converted to the total amount of CO2 per incubation bottle (∑CO2). Excess 13C in the headspace CO2 and DIC (liquid phase) pool was calculated from the change in δ13C-CO2, which is equivalent to a change in the fractional abundance of 13C . The only 13C-organic carbon source in our incubations was polyethylene. Hence, an increase in 13F in the ∑CO2 pool is caused by an excess amount of 13C (13Cex) originating from the added substrate:The rate of polyethylene degradation in assays with polyethylene as the sole carbon source was determined from the increase in the total amount of urements , 60. CO213C (13F ).1\docum13Cex (Δ13Cex) was calculated from the change in δ13C (thus 13F), i.e., the slope of δ13C , washed three times with 1×PBS, and placed in a desiccator at room temperature to dry. Chemical fixation was not performed, thus avoiding dilution of the isotope label. These samples were subsequently measured with nanoSIMS to quantify the 13C labeling of individual R. mucilaginosa cells . In this analysis, R. mucilaginosa cells from the inoculum were used as controls. NanoSIMS data were processed and analyzed using Look@NanoSIMS [Small aliquots (150 µL) of the liquid with the NanoSIMS .13C-labeled polyethylene as the sole organic carbon and energy source to investigate the potential of R. mucilaginosa to degrade and mineralize polyethylene. We used either untreated 13C-polyethylene or 13C-polyethylene that was irradiated prior to incubation with a UV A/B dose corresponding to ~50 and ~125 days of UV irradiation at the sea surface in the subtropical and temperate regions, respectively [We conducted activity assays with ectively .13C-values in the headspace CO2 pool increased linearly by 117‰ over 5 days of incubation when the experiment was conducted with R. mucilaginosa and UV-treated 13C-polyethylene , comprising CO2 in the headspace and DIC in the liquid phase. This, combined with the change in δ 13C-CO2, translates to an excess production of 13C in the carbonate system of the incubations . The 13C excess production is equivalent to the mineralization rate of polyethylene-derived carbon. Based on linear regression analysis, the increase in δ13C in incubations with UV-treated 13C-polyethylene and with R. mucilaginosa was 31.5‰ d–1  of 0.000345 d–1 , and smaller cells with a diameter of ~2 µm  the original inoculum, (ii) after incubation with 13C-labeled polyethylene without and (iii) with UV-treatment. We measured a total of 1144 regions of interest . The small cells in incubations with UV-treated 13C-polyethylene had similar 13C-enrichment with 13F-values of 0.0107. However, the most substantial 13C-enrichment with 13F-values of 0.024 was found in large R. mucilaginosa cells with UV-treated 13C-polyethylene. This was significantly higher than any other cell group, irrespective of the added substrate . Results of the statistical analysis are presented in Supplementary Table In all sample sets, lum Fig. . In contene Fig. . For nanR. mucilaginosa also metabolizes polyethylene-derived carbon in the presence of other, potentially more accessible carbon sources, we additionally incubated R. mucilaginosa in seawater containing 10% of MS medium, with and without UV-treated 13C-polyethylene. NanoSIMS analysis revealed slightly 13C-enriched cells  in incubations where UV-treated 13C-labeled polyethylene was added . No incorporation of the 13C to R. mucilaginosa cells was recorded in incubations where the 13C-polyethylene was not UV-treated.To investigate if R. mucilaginosa. Central to this approach is the utilization of isotopically labeled polyethylene, containing a high degree of 13C instead of 12C, and tracing the polyethylene-derived 13C in the degradation product CO2 and biomass.Microbial degradation, in conjunction with physicochemical processes, may be an important pathway in breaking down plastic litter in the marine environment. Yet, little is known about potential microorganisms degrading marine plastic litter because most quantitative techniques do not allow resolving degradation in the sub-percent range, nor do the experimental setups provide unambiguous proof of microbial utilization of plastics. In this work, we present data from short (<1 week) experimental assays that allowed us to quantitatively measure plastic mineralization rates. In addition, we traced and visualized the assimilation of plastic-derived carbon into individual cells of 2, and we can thus not determine which compounds were utilized by R. mucilaginosa. Nevertheless, the UV-treated 13C-polyethylene was washed and dried before incubation, which likely removed volatile compounds that were generated during the UV treatment. Longer chain degradation compounds and polymers with carbonyl and hydroxyl moieties [13C from isotopically labeled 13C-polyethylene into the terminal oxidation product CO2. Mineralization rates of UV-treated polyethylene with R. mucilaginosa amounted to 0.01% d–1; extrapolated, this translates to degradation rates of 3.8% yr–1 of the initially added polyethylene. These microbially mediated degradation rates are difficult to compare with previously reported rates from literature. Polymer weight loss over time is a frequently used parameter for measuring plastic degradation rates [2. Our methodological approach allows resolving δ13C-CO2 values of ≳1‰ with confidence, which is equivalent to a change in the 13C content in the ∑CO2 pool of ~0.001%. This detection limit is determined by the background CO2 level as well as the added 13C-substrate. For a setup resembling ours , degradation of ~0.002% of the added polyethylene is detectable. This method is consequently orders of magnitude more sensitive than more commonly used methods and also allows measuring plastic degradation rates in much shorter time periods of days to weeks as opposed to months to years.Fungal mineralization of polyethylene-derived carbon occurred in incubations with UV-treated polyethylene, indicating that initial photooxidation enhances fungal plastic degradation in the marine environment. UV-induced photooxidation leads to the formation of carbonyl and hydroxyl moieties in the polymer , 64. Thimoieties , howeveron rates –69. Polyon rates , 70 does13C-labeled biodegradable/compostable plastic poly  (PBAT) and agricultural soil demonstrated incorporation of 13C from the PBAT into fungal hyphae and other unicellular organisms [13C-polyethylene has been used to trace plastic-derived carbon in food webs of a boreal-lake and artificial humic waters [13C enriched cells provide, for the first time, unambiguous proof of the assimilation of polyethylene-derived carbon by a marine-derived fungus. The nanoSIMS measurements show that the incorporation of polyethylene-derived 13C was not homogenous into all fungal cells. We detected two subpopulations of cells, one with significant but low 13C-enrichment and a second population with an extraordinary high enrichment. The cells with higher enrichment were larger in size, thus probably growing or polyploids. Potentially, these cells were in closer proximity to the plastic substrate and incorporated more of the 13C label. The vicinity of the fungal cells to the plastic particles is likely crucial in the degradation process as the plastic substrate is not a soluble compound and is thus not homogenously distributed on a µm scale. Similar to kinetic measurements, we found higher assimilation of polyethylene-derived carbon in R. mucilaginosa cells in incubations with UV-treated polyethylene. Moreover, using nanoSIMS, we could also find a 13C-enrichment in R. mucilaginosa cells in incubation with 13C-polyethylene that was not UV irradiated prior to the incubation experiments. This indicates that the fungus can also degrade the virgin (-UV) polymer, though to a much lesser degree, as shown by the lower biomass 13C-enrichment when compared with our measurements with UV-treated polyethylene. Nevertheless, this will need further confirmation in future studies addressing molecular weight distribution of the polymer and the degree to which it contains short-chain impurities.We measured stable carbon isotope ratios of single fungal cells using nanoSIMS, a technique that, in scanning mode, allows collecting images of isotopic composition with a spatial resolution in the nanometer scale . Previourganisms . Also, lc waters . Our fin13C-polyethylene as potential carbon substrates, we observed label incorporation into R. mucilaginosa cells, although to a much lesser degree. However, this shows that R. mucilaginosa utilizes polyethylene-derived carbon also in the presence of more bioavailable carbon substrates that are easier to metabolize. This consequently implies that R. mucilaginosa could also metabolize polyethylene-derived carbon in the marine environment, where plastic only makes up a fraction of the plethora of available organic matter compounds.Finally, in incubations containing a sucrose-based medium and UV-treated Zalerion maritimum and Alternaria alternata are known to degrade plastic [R. mucilaginosa, is a marine yeast belonging to the Basidiomycota. Yeasts have generally not been described as plastic degraders; however, yeasts are generally ubiquitous throughout fresh, marine, and deep sea environments [–1 in seawater compared to up to 500 cells L–1 in rivers [Rhodotorula is widespread throughout all ecosystems [R. mucilaginosa has been detected in lakes [R. mucilaginosa is common in bioremediation practices, where R. mucilaginosa strains have shown potential as nitrobenzene [R. mucilaginosa has been tested for its ability to remove phenolic compounds from olive mill wastewater [R. mucilaginosa thus seems to be able to break down a variety of hydrocarbon/hydrocarbon-like compounds. Together with our findings, this consequently suggests that Rhodotorula mucilaginosa is a potentially important plastic degrader in a wide range of marine environments. Furthermore, as this yeast has been found in a diversity of aquatic systems globally, it may hence degrade plastics there too.The role and identity of plastic-degrading microbes in the marine environment, specifically fungi is unconstrained. Nevertheless, many different fungi colonize plastic marine debris , 74, 75  plastic , 53. Ourronments . Reporten rivers . The genosystems , includiin lakes , hypersain lakes , arctic in lakes , and thein lakes  and was in lakes . R. muciobenzene  and acryobenzene . Similarstewater , 89. R. Rhodotorula mucilaginosa uses polyethylene-derived carbon for cellular incorporation and energy gain. The ability of R. mucilaginosa to utilize plastic-derived carbon in the presence of other, high-energy-yielding carbon substrates also indicates that fungal plastic degradation can indeed proceed in the natural environment. Our results confirm that initial plastic photooxidation is a key process in making plastic available for subsequent microbial degradation. Most produced and discarded plastic types such as polyethylene and polypropylene float at the ocean surface and will consequently be subjected to photooxidation so that fungal degradation can commence there. At least parts of the vast amounts of plastic litter in the ocean may thus serve as a carbon source for fungi and possibly other microbes, too.Fungi in the marine environment are highly understudied despite their prevalence in the ocean. With the aid of stable isotope assays, we provide unambiguous proof that the fungus Supplementary materialFigure S1Table S1Table S2Table S3


A temperature-controlled electrochemical sensor was constructed based on a composite membrane composed of temperature-sensitive polymer poly (N-isopropylacrylamide) (PNIPAM) and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH). The sensor has good temperature sensitivity and reversibility in detecting Dopamine (DA). At low temperatures, the polymer is stretched to bury the electrically active sites of carbon nanocomposites. Dopamine cannot exchange electrons through the polymer, representing an “OFF” state. On the contrary, in a high-temperature environment, the polymer shrinks to expose electrically active sites and increases the background current. Dopamine can normally carry out redox reactions and generate response currents, indicating the “ON” state. In addition, the sensor has a wide detection range (from 0.5 μM to 150 μM) and low LOD (193 nM). This switch-type sensor provides new avenues for the application of thermosensitive polymers. Dopamine (DA) is an important neurotransmitter produced and released by the central nervous system which plays an important role in many physiological activities, such as emotions, learning, movement, behavior and memory ,2. Imbal2@PS-co-PNIPAM for the temperature-controlled reversible “ON-OFF” electrochemical detection of β-Blocker metoprolol [Stimulus-responsive polymers refer to materials that produce specific responses to molecules or polymers as the external environment  changes, thereby changing the chemical or physical properties of the materials themselves. They have received extensive attention ,16,17,18toprolol . Carbon In this paper, the temperature-sensitive polymer PNIPAM and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) were used to construct an intelligent electrochemical DA sensor with temperature control. Among them, multi-walled carbon nanotubes provide excellent electrochemical and electrocatalytic properties for sensors. The electrochemical process was successfully controlled by adjusting the temperature and the sensitive detection of DA was realized. More importantly, we achieved satisfactory results in drug analysis and the detection of actual human serum samples using this sensor.N-isopropylacrylamide (NIPAM), azodiisobutyronitrile (AIBN), n-hexane, acetone, cyclohexane and potassium chloride were purchased from Sinopharm Chemical Reagent Co., Ltd. . Carboxylated multi-walled carbon nanotubes (with an outer diameter of 20–30 nm and a length of 10–30 microns) were purchased from Chengdu Organic Chemical Co., Ltd. , Chinese Academy of Sciences. Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Tianjin Kemio Reagent Chemical Co., Ltd. . Potassium ferricyanide was purchased from Tianjin Kemio Reagent Chemical Co., Ltd. Dopamine hydrochloride was purchased from Shanghai Meiruier Chemical Reagent Co., Ltd. . The serum was obtained from Huada Forensic Identification Institute in Xixian New Area, Shaanxi Province.We obtained CHI660D electrochemical workstation ), DZ-2BC vacuum-drying oven ), DF-101S heat-collecting constant-temperature heating magnetic stirrer ), 85-2 digital display constant-temperature magnetic stirrer ), and a ZEISS Gemini 300 SEM.Three-electrode system was adopted for electrochemical tests, and glassy carbon electrode, platinum electrode and calomel electrode were purchased from Tianjin Aida Hengsheng Technology Development Co., Ltd. . The ultrapure water used in the experiment was prepared by Shuhuoquan’s YK-RO-B ultrapure water mechanism.v/v = 1:1), wait for the solution to saturate, filter out the insoluble substances, seal it and put it in the refrigerator overnight, and the crystals obtained are NIPAM. Add cyclohexane (18 mL), recrystallized NIPAM (1.25 g) and AIBN (0.012 g) into the reaction flask, stir until completely dissolved, pass N2 for 30 min to remove oxygen, react for 2 h under N2 at 70 °C, stop the reaction, cool down, add acetone to dilute, drop the product into a large amount of n-hexane, generate white precipitate, repeatedly dissolve and precipitate three times, put it into a vacuum-drying oven at 60 °C and dry overnight, and the white product obtained is PNIPAM.PNIPAM was obtained through free-radical polymerization: first, recrystallize the purchased NIPAM to remove the polymerization inhibitor, take an appropriate amount of NIPAM and add it into acetone/n-hexane  and ultrasonically treat the carboxylated multi-walled carbon nanotubes in ultrapure water for 1 h to obtain a uniform dispersion (2 mg·mL−1). Then, combine 10 μL carboxylated multi-walled carbon nanotubes dispersion and 10 μL PNIPAM solution, mix and disperse ultrasonically for 30 min. Then, drop the obtained dispersion onto the surface of the clean GCE. Finally, dry the prepared electrode at room temperature.First, polish the GCE with aluminum oxide powder (1 μm); then, clean ultrasonically in ethanol and HSEM characterization: Use a pipette gun to take the suspended liquid of the sample and drop it on the silicon wafer, dry it naturally in the room, spray gold, and stick the silicon wafer to the sample table with conductive adhesive for testing.−1 PBS (pH 7.0) was carried out by cyclic voltammetry. The CV was carried out at a scan rate of 100 mV·s−1 in a scanning range of −0.2~0.6 V with 2 s standing time. On this basis, DPV was used to further detect DA with a potential range of 0–0.3 V and 2 s standing time. All water solutions were ultrapure water.Electrochemical detection of DA: The traditional three-electrode system was used, in which the modified GCE was used as the working electrode, the platinum electrode as the auxiliary electrode, and the saturated calomel electrode as the reference electrode. The electrochemical determination of DA in 0.1 mol·LEIS analysis: The EIS measurements were carried out by applying the value of the open-circuit potential and recorded over a frequency range of 0.1 Hz to 10 kHz with an ac amplitude of 10 mV.Take 200 μL serum and dilute to 20 mL with 0.1 M PBS (pH = 7). Test with DPV at 40 °C. In addition, a standard addition method was used to calculate the recovery rate.As shown in Electrochemical impedance spectroscopy (EIS) is a crucial electrochemical-measurement technology. It is used to describe changes in the electron-transfer rate at the interface of different modified electrodes and can reflect the conductivity of modified electrode materials. The EIS spectrum is generally composed of semicircles and straight lines: the diameter of the semicircles in the high-frequency area represent the electron-transfer resistance; the straight lines in the low-frequency area represent diffusion control.63−/4− acted as the electroactive probe containing 0.5 M KCl at the frequency range from 0.1 Hz to 10 kHz. The EIS of the GCE, the MWCNTs (COOH)/GCE, PNIPAM/GCE, and PNIPAM/MWCNTs (COOH)/GCE were tested /GCE is very small and its impedance value is close to 0. The impedance value of PNIPAM/MWCNTs(COOH)/GCE (110.7 ohm) is between that of the GCE (1010 ohm) and the MWCNTs (COOH). This shows that, compared with PNIPAM/GCE, PNIPAM/MWCNTs(COOH)/GCE introduced the MWCNTs(COOH), which improved the conductivity of the composite membrane and reduced the obstruction in the electron transmission. This also shows that the temperature-sensitive polymer with poor conductivity and the MWCNTs(COOH) with excellent conductivity have a very good synergy, which was proof that the two successfully formed composite materials with a good electrochemical performance.As shown in e tested A. In orde tested C. In theAs shown in This shows that the redox process of DA on the composite electrode is controlled by adsorption.θ. The linear equation with pH values is:θ are −66.63, −59.77 and −63.2 mV/pH, respectively. According to the relationship between pH values and peak potential [The slopes of Epa, Epc and Eotential , the numotential , the feaIn order to investigate the temperature-response characteristics of the composite modified electrode, the electrochemical response of DA on PNIPAM/MWCNTs (COOH)/GCE at different temperatures was studied by cyclic voltammetry. It can be seen from In order to further explore the reasons why PNIPAM/MWCNTs/GCE detects the temperature-response switch property of DA, the temperature-sensitive behavior of MWCNTs (COOH) was investigated and the MWCNTs (COOH)/GCE were prepared. It can be seen from DPV was used to investigate the relationship between DA oxidation-peak currents and concentration at a solution temperature of 40 °C. As can be seen in The detection limit is 0.193 μM. Compared with previously reported detection methods , these mAs can be seen in +, Cl−, Na+, Ca2+, Glucose, ascorbic acid and uric acid. As shown in DPV was used to discuss the influence of interfering substances on DA detection. We added DA to 0.1M PBS solution (DA concentration: 30 μM), before adding more than 5 times’ worth of other substances, including KIn order to explore whether the composite modified electrode can be applied to the detection of actual samples, the recovery rate of DA in serum was calculated using a standard addition method. The experiment found that the recovery rate was between 98.7 and 104.4 , indicatIn this paper, a temperature-reversible switching electrochemical sensor based on the thermosensitive polymer PNIPAM and MWCNTs (COOH) was constructed for the detection of dopamine. The experimental results show that the sensor can trigger the DA temperature-switch control, which is very sensitive and completely reversible. In addition, the sensor has high stability and a good detection performance for DA and obtained good detection and recovery results in human serum samples. This new switch-type sensor also provides new avenues for the application of thermosensitive polymers. Due to the poor conductivity of polymers, we will focus on developing polymers with better conductivity and heat sensitivity and establish ultra-sensitive and ultra-low LODs for temperature-controlled electrochemical sensors.


Irisin (Ir) is an adipomyokine formed from fibronectin type III domain-containing protein 5 (FNDC5), which can be found in various cancer tissues. Additionally, FNDC5/Ir is suspected of inhibiting the epithelial-mesenchymal transition (EMT) process. This relationship has been poorly studied for breast cancer (BC). The ultrastructural cellular localizations of FNDC5/Ir were examined in BC tissues and BC cell lines. Furthermore, we compared serum levels of Ir with FNDC5/Ir expression in BC tissues. The aim of this study was to examine the levels of EMT markers, such as E-cadherin, N-cadherin, SNAIL, SLUG, and TWIST, and to compare their expression levels with FNDC5/Ir in BC tissues. Tissue microarrays with 541 BC samples were used to perform immunohistochemical reactions. Serum levels of Ir were assessed in 77 BC patients. We investigated FNDC5/Ir expression and ultrastructural localization in MCF-7, MDA-MB-231, and MDA-MB-468 BC cell lines and in the normal breast cell line (Me16c), which was used as the control. FNDC5/Ir was present in BC cell cytoplasm and tumor fibroblasts. FNDC5/Ir expression levels in BC cell lines were higher compared to those in the normal breast cell line. Serum Ir levels did not correlate with FNDC5/Ir expression in BC tissues but were associated with lymph node metastasis (N) and histological grade (G). We found that FNDC5/Ir correlated moderately with E-cadherin and SNAIL. Higher Ir serum level is associated with lymph node metastasis and increased grade of malignancy. FNDC5/Ir expression is associated with E-cadherin expression level. Breast cancer (BC) is the most prevalent cancer in women, and it is considered the most common cause of death in cancer patients . TreatmeIrisin (Ir) is a transmembrane protein  that wasIt was also suggested that Ir inhibited the proliferation, migration, and EMT of cancer cells by affecting the PI3K/AKT/SNAIL pathway . Such reEMT is a process during which an epithelial cell (expression of E-cadherin) transforms into a mesenchymal cell (expression of N-cadherin). The cell loses structural connections with other cells and loses its polarity. E-cadherin is one of the transmembrane proteins and also one of the most significant epithelial markers. It participates in the formation of the cytoskeleton and is responsible for maintaining normal cell polarity . It playThe relationships between the level of Ir expression, its secretion, EMT, and cancer progression are very complex and may result in opposite effects under different conditions. Therefore, further research is warranted to explain the differences in such relationships. In this study, the level and localization of the above markers in the tissues of BC patients were determined. These markers have not yet been compared with the level of FNDC5/Ir expression in such a large cohort. We also examined the localization and expression level of FNDC5/Ir in cell lines. Furthermore, we compared serum Ir levels with FNDC5/Ir expression levels in BC tissue. To the best of our knowledge, it has never been reported before. Moreover, the ultrastructural cellular localization of Ir in BC tissues was assessed for the first time. Additionally, due to the differences in the research results related to the effect of Ir on the progression of cancer in different types, the aim of this study was to examine the levels of EMT markers, such as E-cadherin, N-cadherin, SNAIL, SLUG, and TWIST, and to compare their expression levels with the level of FNDC5/Ir expression in BC.To investigate the relationship between FNDC5/Ir expression level and EMT transition, correlations with EMT markers, such as E-cadherin and N-cadherin, SNAIL, SLUG, and TWIST, were evaluated. We compared the previously obtained results of FNDC5/Ir expression in BC tissues  with thep < 0.0001)   (p < 0.0001) and a weak positive correlation for nuclear  expression of TWIST  A. The ex 0.0001) B. Cytoplctively) C,D. In cctively) E,F. We oof TWIST G,H.Serum Ir concentrations were evaluated by ELISA. The association of the results of serum Ir levels in patients with the clinicopathological data, as well as with the results of the FNDC5/Ir expression level in the BC tissue were investigated.p = 0.0055, p = 0.0072, respectively) (p = 0.0166)  B. We obs 0.0166) C. We fou 0.0166) D. Table FNDC5 mRNA in cells using RT-PCR.Some examinations were conducted on BC cell lines . The normal breast cell line (Me16c) was used as the control. We estimated the FNDC5/Ir expression level in cells by measuring the level of immunofluorescence using confocal microscopy. In addition, we examined the expression level of FNDC5 mRNA expression levels in normal breast cells (Me16c) were significantly lower (mean 1.03 ± 0.05 SD) compared to the levels found in the cells of all BC lines . In addition, the expression levels were significantly different between BC cell lines. The highest level of expression of the FNDC5 gene was observed in MDA-MB-468 cells. The difference between the FNDC5 mRNA levels in MCF-7 and MDA-MB-231 lines was statistically significant  (ctively) A.p < 0.0001; MDA-MB-231 mean 37.81 ± 20.47 SD, p = 0.0327; MDA-MB-468 mean 74.13 ± 15.40 SD, p < 0.0001). The difference between the FNDC5/Ir levels in MDA-MB-231 and MCF-7, MDA-MB-468 lines was statistically significant   showed that the level of FNDC5/Ir expression in normal breast cells (Me16c) was significantly lower (mean 18.54 ± 4.50 SD) compared to the level in the cells of all BC lines  B. FNDC5/Immunolocalization of FNDC5/Ir at the ultrastructural level was performed after the application of the post-embedding method. First observations were made on invasive ductal carcinoma (IDC) tumors at different stages of malignancy (G2 and G3) . In bothThis study is a continuation of our previous studies evaluating the expression level of FNDC5/Ir in BC tissue . In thisSNAI1, SNAI2, and TWIST1, was also observed in non-spindle cell lines. Additionally, the positive correlation between FNDC5/Ir and E-cadherin did not preclude the occurrence of EMT in BC cells, which is in line with the findings of Hollestelle et al. This indicates a high complexity of interactions between E-cadherin repressors.For metastasis to occur, the EMT process must occur in the cancer cells . The mosIn our study, we observed weak and moderate positive correlations between SLUG, TWIST, SNAIL, and FNDC5/Ir levels. Such correlations were found in the cytoplasm and the nucleus of BC cells. SLUG, TWIST, and SNAIL are transcription factors that induce EMT in the cell by modifying the expression of many proteins, including the inhibition of E-cadherin expression . The posFNDC5/Ir may not only influence the levels of the transcription factors  in the tissue but also their activity in the cell. Wang et al.  indicateThe high complexity of the effect of FNDC5/Ir on EMT was demonstrated by various studies ,16,17. AIn our study, we also analyzed and compared serum and tumor levels of Ir from BC patients. We noticed a tendency for a positive relationship between the expression level of FNDC5/Ir in BC tissue and serum concentrations of Ir in patients. However, this relationship was not statistically significant. The lack of statistical significance may result from various reasons. First, newly synthesized FNDC5 in the cell must undergo a complex pathway before it is converted to glycosylated Ir and released into plasma. The release of Ir into the serum is still not fully understood. The factors affecting changes in plasma concentrations of Ir are still not clear. Such factors may include gender, age, the level of physical activity, or the stage of the menstrual cycle . In addiHowever, when the relationship between plasma Ir levels and the clinicopathological data was analyzed, we observed significantly higher concentrations of Ir in patients with many lymph node metastases (N2) compared to patients without metastases (N0). So far, several studies on evaluating plasma concentrations of Ir in BC patients have been conducted. However, only two of them investigated the relationship between serum levels of Ir and the clinicopathological data of the patients. Our results are consistent with those presented by Provatopoulou et al. . They alIn the immunogold study, we confirmed that FNDC5/Ir was present in the cytoplasm of BC cells in tissue as well as in cell lines. FNDC5/Ir was observed in the mitochondria, cell membranes, near the intermediate filaments, and the rough endoplasmic reticulum. This confirms FNDC5/Ir expression observed by IHC and IF. Additionally, we observed Ir in the extracellular matrix and the cells of stromal fibroblasts. Nowinska et al.  also detFNDC5 in the MDA-MB-468 cell line which is the most aggressive of the tested cell lines because of the triple-negative receptor status . Cancer cells show accelerated metabolism associated with the intensification of glycolysis or mitochondrial biogenesis [We also conducted research on BC cell lines. We examined the FNDC5 expression level and the FNDC5/Ir protein level. Studies on an in vitro model confirmed a higher level of FNDC5/Ir expression in BC tissues compared to NMBD tissues, which we previously observed . As in togenesis . The aboogenesis . PGC1α iogenesis .In conclusion, the expression level of FNDC5/Ir in BC cell lines was higher than in normal BC epithelial cells. Ultrastructural studies showed the presence of FNDC5/Ir in the cytoplasmic structures of BC tumor cells and stromal fibroblasts. Higher Ir serum levels are associated with lymph node metastasis and a higher grade of malignancy. However, serum Ir levels in patients did not reflect its levels in BC tumor cells. Serum Ir levels in patients might also be affected by its secretion by other tissues, e.g., muscle and adipose. Further research is warranted to clarify the mechanisms of Ir release from cells to plasma and the regulation of this process. Additionally, FNDC5/Ir expression is associated with the expression level of E-cadherin and correlates with EMT marker expression in BC tissues. However, the results are contradictory and need to be confirmed in further studies. Therefore, further studies are warranted to explain the mechanism by which FNDC5/Ir influences E-cadherin upregulation and EMT transition.The study was conducted on archival material consisting of 541 tumor tissues .Molecular biology studies were conducted using the adherent BC cell lines  obtained from The American Type Culture Collection . The Me16c cell line was used as the control. MCF-7 cells were cultured in an EMEM medium . Leibovitz’s L-15 Medium  was used to culture MDA-MB-231 and MDA-MB-468. Me16c was cultured in MEBM medium  supplemented with insulin, hydrocortisone, bovine pituitary extract (BPE), and the human epidermal growth factor (hEGF). All media were supplemented with 10% fetal bovine serum (FBS) , 1% penicillin/streptomycin , and L-glutamine (Merck). The HERA cell incubator  was used to maintain constant cell culture conditions  were performed on 541 BC and 61 NMBD sections. Slides with the whole BC or NMBD tissue sections were stained with hematoxylin and eosin and scanned with the Pannoramic Midi II  histological scanner. Three demonstrative sites with cancer were selected by the Pannoramic Viewer . Subsequently, the selected cancer sites were transferred with a core of 1.5 mm to the tissue arrays using the TMA Grand Master (3D HISTECH Ltd.).Immunohistochemical reactions were performed on each TMA section. Deparaffinization, hydration, and thermal epitope demasking were completed using a low pH Target Retrieval Solution  for 20 min at 97 °C in a Dako PT Link  apparatus. The expression of proteins was detected by specific primary antibodies, i.e., polyclonal rabbit: anti-irisin/FNDC5  and anti-SNAIL , monoclonal mouse: anti-E-cadherin antibody , anti-N-cadherin , anti-SLUG , and monoclonal mouse anti-TWIST . DAKO Autostainer Link48 (Dako) automated system and an EnVision FLEX kit (Dako) were used to visualize the antigens.At least two independent pathologists-researchers conducted the evaluation of the IHC reactions at ×200 magnification. The assessment was performed using a BX41 light microscope  coupled with a visual circuit and the Cell D (Olympus) software. The nuclear expression levels of SNAIL, TWIST, and SLUG were determined using a five-point evaluation scale (% of nuclear expression)  . The finFNDC5 expression  in the cell lines. The RQ Manager 1.2 software (Applied Biosystems) was used for the analysis. The results were standardized by the reference gene for β-actin expression . Changes in the mRNA FNDC5 level in BC cells were assessed in relation to normal cells. RT-PCR was repeated three times.RT-PCR reactions were performed for BC cell lines and the control cell line. The RNeasy Mini Kit (Qiagen) was used for RNA isolation. The High-Capacity cDNA Reverse Transcription Kit  with RNase Inhibitor (Applied Biosystems) was used to perform the reverse transcription reaction. The 7900HT Fast Real-Time PCR System (Applied Biosystems) and the relative quantification (RQ) method were used to analyze mRNA 4 cells/mL suspension of cells was instilled into each well of Millicell EZ 8-well glass slides (Merck). Microcultures with cells were incubated at 37 °C for 24 h. Then, the cells were fixed using 4% formaldehyde. Subsequently, the fixed cells were incubated with the specific polyclonal rabbit anti-irisin/FNDC5 antibody  at 4 °C overnight. The slides with the fixed cells were rinsed and incubated for 1 h with donkey anti-rabbit secondary AlexaFluor 568 conjugated antibody . The secondary antibody was diluted in a background-reducing reagent . The Prolong DAPI Mounting Medium (Invitrogen) was used to stain the cell nucleus and mount the slides. The observations were made at ×600 magnification using Olympus Fluoview FV3000 confocal microscopy coupled with CellSense (Olympus) software.For 24-h microculture, 600 μL of 20 × 10The enzyme-linked immunosorbent assay (ELISA) tests were performed on serum samples from BC patients. Serum samples were obtained from venous blood after centrifugation at 3000 rpm for 10 min and were preserved at −80 °C for further analysis. Serum Ir levels were examined using commercially available ELISA recombinant irisin kit test Irisin, Recombinant Elisa kit . The ELISA tests were performed according to the manufacturer’s protocol. For each sample, 50 μL of serum was used. The results were verified by microplate reader ELX-800 . The examination was performed in two repetitions.w/v) solution of PBS and osmium tetroxide . Following washing with PBS (3 × 5 min), dehydration of the specimens was completed in a graded series of ethanol . The samples were incubated with a mixture of 99.8% ethanol and LR White resin   combined in appropriate proportions, i.e., 2:1 (20 min), 1:1 (1 h), and 1:2 (1 h), respectively. Next, the material labeled with the corresponding number was placed at the bottom of the gelatine capsule , embedded in pure resin, and underwent the hardening process in the laboratory incubator (55 °C for 48 h). Resin blocks were sectioned using the RMC ultramicrotome  into 700-nm-thick semithin sections. These sections, which were mounted onto the slide on the heating plate (at 100 °C), were stained with 1% aqueous toluidine blue  mixed with sodium carbonate . This staining is dedicated to semithin sections and allows obtaining different intensities of tissue fragments, which facilitates trimming of the area, cancer cells, and stroma with the exclusion of fat tissue. After trimming, serial 80-nm-thick ultrathin sections were obtained using the ultra-diamond knife and placed on the grids . The immunogold reaction was performed to trace the distribution of Ir. All steps were performed in the Petri dish. The first step was based on blocking residual aldehyde groups by placing the sections in 0.02 M glycine solution  in PBS for 10 min. Next, the samples were immersed in triton X-100  to increase membrane permeability and then washed with PBS . The sections were subsequently blocked for 1 h in a 1% bovine serum albumin  to inhibit the nonspecific antigen-binding sites and quickly washed with PBS.After the application of TEM, the subcellular localization of Ir was examined in two types of samples. Breast tumors sections were cut into small pieces and in vitro culture of the MDA-MB-468 cell line representing metastatic adenocarcinoma of the breast was immersed in a 4% solution of PBS and paraformaldehyde  and fixed overnight at 4 °C (for tumors) or 25 min at room temperature (for the cell line). Subsequently, the fixed cells were gently removed with a cell scraper and placed in conical tubes (15 mL). After three cycles of centrifugation (1800 rpm for 8 min), the fixative was washed with PBS and distilled water. The cell pellets were entrapped in a fibrin clot formed from bovine thrombin  and fibrinogen . To preserve cellular ultrastructure, each sample was post-fixed for 7 min in a 0.25%  and then anti-rabbit secondary antibody tagged with colloidal gold particles  for 1 h in a dark chamber. The steps were as follows: grids were rinsed in PBS and distilled water, post-fixed in 1% glutaraldehyde , and post-stained with the UranyLess solution and Reynolds lead citrate 3%  to preserve the ultrastructure quality and washed thoroughly in distilled water. JEM-1011  transmission electron microscope operating at the accelerating voltage of 80 kV was used. Electronograms were obtained using the TEM imaging platform (iTEM1233) equipped with a Morada Camera .The distribution of the results was checked by the Kolmogorov–Smirnov test. The differences in the serum levels of markers or tissue expression in BC and NMBD and their association with the clinicopathological factors were analyzed by the Kruskal–Wallis test or the Mann–Whitney U test. Correlations between Ir and E-cadherin, N-cadherin, SNAIL, and SLUG proteins were evaluated by the Spearman rank test. The unpaired t-test was used to assess the differences between the Ir level in BC and the control cell lines. Statistical analysis was performed using Prism 5.0 . P values below 0.05 were considered statistically significant.


Long non-coding RNA (lncRNA) plays a vital role in tumor proliferation, migration, and treatment. Since it is challenging to standardize the gene expression levels detected by different platforms, the signatures composed of many immune-related single lncRNAs are still inaccurate. Utilizing a gene pair formed of two immune-related lncRNAs and strategically assigning values can effectively meet the demand for a higher-accuracy dual biomarker combination.Co-expression and differential expression analyses were performed on immune genes and lncRNAs data from The Cancer Genome Atlas and the ImmPort database to obtain differentially expressed immune-related lncRNAs for pairwise pairing. The prognostic-related differentially expressed immune-related lncRNAs (PR-DE-irlncRNAs) pairs were then identified by univariate Cox regression and used for lasso regression to construct a prognostic model. Various methods were used to validate the predictive prognostic performance of the model. Additionally, we explored the potential guiding value of the model in immunotherapy and chemotherapy and constructed a nomogram suitable for efficient prognosis prediction. Mechanistic exploration of anti-tumor immunity and mutational perspectives are also included. We also analyzed the correlation between the model and immune checkpoint inhibitors (ICIs)-related, N6-methyadenosine (m6A)-related, and multidrug resistance genes.We used a total of 20 pairs of PR-DE-irlncRNAs to create a prognosis model. Quantitative real-time polymerase chain reaction experiments further verified the abnormal expression of 11 lncRNAs in HNSCC cells. Various methods have confirmed the excellent performance of the model in predicting patient prognosis. We reasoned that lncRNAs/TP53 mutation might play a positive/negative anti-tumor role through the immune system by multi-perspective analyses. Finally, it was found that the prognostic model was closely related to immunotherapy and chemotherapy as well as the expression of ICIs/m6A/multidrug resistance-related genes.The prognostic model performs excellently in predicting the prognosis of patients and provides the potential value of practical guidance for treatment.The online version contains supplementary material available at 10.1186/s12957-023-03066-x. In advanced stages of head and neck squamous cell carcinoma (HNSCC), an aggressive malignancy, mortality, and morbidity are high . With apLncRNAs have been demonstrated to regulate cancer progression through immune regulation, mainly by changing the immune microenvironment. Xiong et al. found that with the upregulation of lncRNA-POU3F3 expression in cancer-related cells, circulating regulatory T cells (Tregs) increased in gastric cancer patients . In vitrWe aspire to obtain differentially expressed immune-related lncRNAs (DE-irlncRNAs) by combining the HNSCC RNA sequencing data obtained from the TCGA database and the immune-related genes obtained from the ImmPort database. The effective paired DE-irlncRNAs were then screened to find the prognostic-related DE-irlncRNAs pairs (PR-DE-irlncRNAs pairs). Following this, a prognostic model was designed based on 20 pairs of PR-DE-irlncRNAs, and the sample risk score was calculated. We performed Risk plots, Receiver Operating Characteristic (ROC) curves, Kaplan–Meier (K-M) curves, Cox regression analysis, and clinical features subgroup analysis to verify the accuracy of the model’s predictive ability. In parallel, we investigated their possible mechanisms of action in HNSCC by gene set enrichment analysis (GSEA), tumor immune microenvironment (TIME) analysis, and mutation analysis. Finally, to determine whether prognostic models can be applied to treatment guidance and prognosis evaluation for patients with HNSCC, immunophenotypic score (IPS) analysis, drug sensitivity analysis, ICIs/m6A/multidrug resistance-related genes analysis and nomogram were used.The transcription component data of 502 HNSCC samples and 44 adjacent normal tissues were downloaded from The Cancer Genome Atlas (TCGA) database. Tumor and normal oral tissue were not paired. Simultaneously, the clinical data  were obtained in an identical manner. For the following analysis, 2483 immune-related genes (IRGs) were extracted from the ImmPort database.p value < 0.001. The expression values of 1718 IRGs and 14,086 lncRNAs extracted from the expression matrix of TCGA were used to filter irlncRNAs. The R package “limma” was then applied to the differential expression of irlncRNAs between 504 HNSCC samples and 44 normal tissues to identify the DE-irlncRNAs according to the filtering condition that was set to |log2FC (fold-change)| >1 and FDR <0.001 [i and lncRNA j. The K equals 1 if the expression level of lncRNA i is higher than that of lncRNA j, and the reverse is K = 0. These values of lncRNA pairs K were used for further filtering for effectively matched lncRNA pairs. The lncRNA pairs K defined as 0 or 1 could be identified as effectively matched lncRNA pairs for the subsequent analysis on the condition that their numbers accounted for more than 20% and less than 80% of all samples.By co-expression analysis, the threshold was set to the correlation coefficient >0.5 and R <0.001 . The lncp value <0.001); 499 samples with complete OS data and defined values of PR-DE-irlncRNAs pairs were randomly assigned to the training set (n = 300) and the validation set (n = 199) at a ratio of 6:4 for the subsequent analysis. To screen out highly correlated PR-DE-irlncRNAs pairs, the lasso regression was used to analyze defined values of 97 PR-DE-irlncRNAs pairs, which could minimize the risk of overfitting for screening signatures. Finally, the optimal penalty parameter (λ) determined by the minimum 10-fold cross-validation was employed to construct a prognostic model based on 20 PR-DE-irlncRNAs pairs. After optimizing the model, the formula for calculating the risk score of the sample was listed as follows:After integration, to filter for PR-DE-irlncRNAs pairs, we analyzed the data of HNSCC samples with complete overall survival (OS) and effectively matched lncRNA pairs by univariate Cox analysis  between the high-risk and the low-risk groups to investigate the biological functions and pathways related to the prognostic model. We set the threshold to |log2FC| ≥1 and FDR <0.05 to filter out DEGs. In addition to utilizing the Gene Ontology (GO) for investigating possible biological functions, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was also used to identify possible pathways involved in DEGs. The research was conducted by the R package “clusterProfiler,” “org.Hs.eg.db,” and “enrichplot,” with corresponding results displayed in bar plots and bubble graphs .p <0.05) were demonstrated. Mann–Whitney U test analysis was utilized to compare the content of immune infiltrating cells between high-risk and low-risk groups, with final results displayed in violin plots. The R packages “ggplot2,” “scales,” “ggtext,” and “vioplot” were used in the aforementioned analyses [U test analyses were also used during this process.To evaluate the correlation between immune cells/stromal cells and risk score, the R package “estimate” was used to calculate immune and stromal cell scores for each sample. We then compared the difference in immune and stromal cell scores of patients between the high-risk and low-risk groups. Moreover, Spearman correlation analysis was used to analyze the correlation between immune/stromal cells score and risk score. To further explore the relationship between tumor-infiltrating immune cells and the prognostic model, we used the immune infiltrating cell content of each HNSCC sample calculated by the five most advanced algorithms  from the TIMER 2.0 database (timer.comp-genomics.org). We applied Spearman correlation analysis to evaluate the connection between risk score and immune infiltrating cells and used the point graph to visualize the correlation results. Only the results with the significant correlation  and MRP3 (ABCC3), which probed their correlation with the prognostic model. R packages “ggplot2” and “reshape2” were included in the analysis .We downloaded the IPS from the Atlas of Cancer Immunity (TCIA) database. IPS of the patients was obtained by evaluating the gene expression of four cell types  that determined immunogenicity . SpearmaA multivariate Cox regression model consisted of risk scores and clinical factors that were significantly related to prognosis, as filtered out by univariate Cox regression, in order to create a more clinically appropriate quantitative tool for predicting the 1-, 2-, and 3-year OS. The final result was presented as Nomogram, while the calibration curve was used to estimate the accuracy of survival prediction. In addition, we employed the multi-factor ROC curve to verify the accuracy of the Nomogram and optimality in predicting the 1-, 2-, and 3-year OS. In this process, the R packages “rms,” “survival,” and “survivalROC” were used.We purchased human oral squamous cell carcinoma cells  from Shanghai Anwei Biotechnology Co., Ltd. In addition, we purchased normal oral gingival epithelial cell line (HEG) from Shanghai Baiye Biotechnology Center. We cultured these three cells in complete DMEM  or DMEM/F12  containing 10% fetal bovine serum  and 1% penicillin-streptomycin liquid . All cells were cultured at 37 °C in 5% CO2’s humidified incubator.−ΔΔCt method, we compared differences in the expression levels of 15 lncRNAs between cal27/scc9 and HEG cells.The 20 PR-DE-irlncRNA pairs used for modeling comprised of 35 lncRNAs. We endeavored to design primers for these lncRNAs required for QRT-PCR  experiments. Ultimately, we only succeeded in designing primers for 14 lncRNAs that can be used effectively in QRT-PCR. Table t-test or Mann–Whitney U test was utilized to compare continuous variables. At the same time, the chi-square test or Fisher exact test was used to compare categorical variables. DE-irlncRNAs pairs related to prognosis, which univariate Cox regression analysis confirmed, were used to screen out the optimal genes to construct the prognostic model by lasso regression. Multivariate Cox regression was used to construct the Nomogram. The study employed the K-M curve with the log-rank test to compare survival between different groups, while the ROC curve was used to evaluate the prognostic predictive ability of each factor. The univariate and multivariate Cox regression analyses were applied to evaluate the independent predictive value of risk scores. Spearman or Pearson was used for analyzing the correlation between two variables. We performed all analyses in our study in R programming language (version 4.0.3), Perl (version 5.32.1001), and SPSS Statistics software 22. All statistical p values were two-tailed, and the results identified as statistically significant had the threshold of the p value <0.05 or FDR <0.05.According to the distribution characteristics, Student’s In Fig. λ  shown in Fig. Through integrating mRNA expression and clinical data of HNSCC patients, we acquired the clinical features of 499 HNSCC samples shown in Table λ Fig. S. Table 3p < 0.05). After adjusting other clinical confounding factors by multivariate Cox regression analysis, the risk scores of the three sets still remained significantly correlated with survival (p < 0.05), thereby indicating that the risk score could serve as an independent predictor of prognosis.The distribution of risk score, survival time, and status of three sets was shown in Fig. p < 0.001), age (p < 0.05), T (p < 0.01), and N (p < 0.05) and the risk score calculated by the prognostic model. Furthermore, higher mortality observed in samples with the advanced T stage and N stage was more concentrated in the high-risk group. It was noteworthy that many clinically valuable results were found in the further difference analysis of the relationship between risk score and different clinical characteristics groups .The distribution of clinical features of all samples with the increase of risk score was shown in Fig. ups Fig. B–H. Highups Fig. B–C. Sampups Fig. D. Figureups Fig. F. Furtheups Fig. A-G, 5I–Pups Fig. H. Additip < 0.001)     Fig. A. Higher01) Fig. B, which 01) Fig. C–D. To e01) Fig. E–F. In tund Fig. G. Besidep < 0.001)    Fig. E and a w05) Fig. F were di05) Fig. G. Conver05) Fig. G. At the05) Fig. H.Fig. 6MWe evaluated the correlation between 46 ICIs-related genes and risk scores , cellular component (CC), and molecular functions (MF) significantly related to these genes were enriched and displayed in Fig. SIn OSCC cells scc9 or cal27), the relative RNA expression levels of AC098487.1, MIR924HG, RUSC1-AS1, LINC00205, SNHG25, KDM4A-AS1, AL354733.3, HOXC-AS1, AL390719.2, and AL133243.2 were higher . Among them, higher scores of checkpoint, cytolytic activity, HLA, inflammation-promoting, T cell co-stimulation, and type II IFN response were observed in the low-risk group (p < 0.05). CCR 5 could recruit MDSC to tumors closely related to tumor immunity [In addition, the scores of CCR, APC co-stimulation , type IIimmunity . Type IIimmunity . Previouimmunity . Tumor mimmunity , which mAmong the mutations in HNSCC, TP53 mutation was found to be the most common mutation in both high-risk and low-risk groups. It was well-established that the number of p53-regulated lncRNA increased rapidly, indicating their widespread involvement downstream of p53 activation . TranscrIn addition, we found that the expression of PDL1 (CD274) decreased significantly in the TP53 mutation group, which may lead to an increase in the tumor and cancer stem cell phenotype in cholangiocarcinoma. At the same time, it was found that low CD274 had high tumor initiation potential . At presMore and more evidence showed that m6A RNA methylation played a crucial role in tumorigenesis. m6A modification of some genes may result in changes in mRNA behavior and expression, thus accelerating tumor development, whereas the lack of m6A modification of other genes might still lead to tumor progression . We studOur study observed that IPS-CTLA4 and IPS-PD1 + CTLA4 were negatively correlated with a risk score. Some experiments showed that in HNSCC, the scores of IPS with CTLA4 blocker, IPS with CTLA4, and PD1/PDL1/PdL2 blocker in the low-risk group were significantly higher than those in the high-risk group, which was consistent with our experimental results . This meIt is worth acknowledging that our research had certain shortcomings and limitations. Firstly, due to the lack of data sets containing complete lncRNA and mRNA transcription data in other shared databases, we only relied on a distinct data set from TCGA to build and validate our model, which may lead to randomness in the results. The lack of an external validation set posed a challenge to the reliability of model performance. To compensate for this limitation, three data sets obtained by randomly splitting the TCGA data set were used to thoroughly verify the model’s performance. In addition, it is noteworthy that the limited number of normal samples also poses challenges to the accuracy of differential analysis results. Furthermore, due to the large number of lncRNAs in the model and limited experimental conditions, it was challenging for us to perform qRT-PCR to verify the differential expression of these lncRNAs. However, we obtained many valuable conclusions through multi-perspective analysis, regarding both clinical application and the underlying mechanism of HNSCC progress. Nevertheless, these conclusions need to be further verified in subsequent experimental studies.The prognosis model included a total of 20 PR-DE-irlncRNAs pairs. Various methods have verified that the risk score calculated from 20 pairs of PR-DE-irlncRNAs has an excellent prognostic capability to predict the prognosis of patients with HNSCC. The prognostic model also performs well in the clinical risk classification and treatment guidance of HNSCC patients.Additional file 1: Fig. S1. The minimum 10-fold cross-validation determined the optimal penalty parameter (λ).Additional file 2: Fig. S2. Survival correlation analysis in different subtypes of each clinical feature. The changes of survival probability with time in different subtypes of different clinical characteristics in high-risk and low-risk groups.Additional file 3: Fig. S3. The six most advanced algorithms calculate the comparison between high and low-risk groups in different immune cell contents. (A) CIBERSORT and ABS, (B) XCELL, (C) TIMER, QUANTISEQ, and EPIC.Additional file 4: Fig. S4. The relevance analysis between risk score and ICIs-related genes/m6a-related / multidrug resistance genes' expression level and the comparison of these parameters in different risk groups.  The expression level of ICIs-related genes.  The expression level of m6a-related genes.  The expression level of ABCC1.  The expression level of ABCC3.Additional file 5: Fig. S5. The multi-factor ROC curves were used to confirm the Nomogram with the optimal predictive performance. 1-, 2-, and 3-years of multi-factor ROC curves based on three sets: (A-C) The training set. (D-F) The test set. (G-I) The whole set.Additional file 6: Fig. S6. Results of GO and KEGG enrichment analysis. (A) BPs, CCs, and MFs that are strikingly enriched by GO. (B) Pathways are strikingly enriched by KEGG. Different colors of circles and rectangles represent different significances. The size of the circle corresponds to the different ratios of DEGs enriched by each function to the total number of DEGs.


Single-photon emission computed tomography (SPECT) is an important tool for myocardial perfusion imaging (MPI). Mechanical collimators cause the resolution-sensitivity trade-off in the existing cardiac SPECT systems, which hinders fast cardiac scan capability. In this work, we propose a novel collimator-less cardiac SPECT system with interspaced mosaic-patterned scintillators, aiming to significantly improve sensitivity and reduce scan time without trading-off image resolution.We propose to assemble a collimator-less cardiac SPECT with 7 mosaic-patterned detector modules forming a half-ring geometry. The detector module consists of 10 blocks, each of which is assembled with 768 sparsely distributed scintillators with a size of 1.68 mm × 1.68 mm × 20 mm, forming a mosaic pattern in the trans-axial direction. Each scintillator bar contains 5 GAGG(Ce) scintillators and 5 optical-guide elements, forming a mosaic pattern in the axial direction. In the Monte Carlo simulations, the in-plane resolution and axial resolution are evaluated using a hot-rod phantom and 5 disk phantoms, respectively. We simulate a cardiac phantom that is placed in a water-filled cylinder and evaluate the image performance with different data acquisition time. We perform image reconstruction with the expectation–maximization algorithm using system matrices derived from the simulation of a uniform cylindrical source filling the field-of-view (FOV). Besides, a 2-D prototype system is designed to demonstrate the feasibility of the collimator-less imaging concept.In the simulation system, the sensitivity is 16.31% ± 8.85% in a 180 mm (Φ) × 100  mm (L) FOV. The 6-mm rods in the hot rod phantom and the 5-mm disks in the disk phantom are clearly separable. Satisfactory MPI image quality is achieved in the cardiac phantom study with an acquisition time of 30 s. In prototype experiments, the point sources with an 8 mm center-to-center distance are clearly separable at different positions across the FOV.The study reveals a promising approach to high-sensitivity SPECT imaging without a heavy-metal collimator. In cardiac imaging, this approach opens the way to a very fast cardiac scan with good resolution. Further works are ongoing to build a practical 3-D imaging system based on the existing design. The typanalysis , 4.99mTc-sestamibi, a most widely used tracer in MPI imaging  without the resolution recovery techniques according to the National Electrical Manufactures Association NU-12001 protocol, where the standard orbit radius for dual-head SPECT is 15 cm . The typ imaging . The acq imaging . A new d imaging , 12, 13. imaging . CardiAr imaging , 15. Oth imaging , and som imaging , 25. Bes imaging . However imaging , 28.Fast scanning is critical for cardiac imaging. However, existing cardiac SPECT scanners require a certain mechanical collimator to form projection, which causes substantial photon loss and tempers sensitivity. Compared to the conventional parallel-hole collimator, although focusing collimators such as multi-pinhole, slit-slat, or cardiac fan-beam collimators effectively improves the resolution-sensitivity trade-off in the heart FOV, the inevitably dramatic photon loss on the mechanical collimators still lead to an overall low sensitivity for the imaging system and strongly limit the fast imaging capability.Compton camera is one collimator-less imaging method that can improve system sensitivity significantly. Compton-camera has been used in small animal imaging studies with millimeter-level resolution . HoweverRecently, our lab has proposed a novel collimator-less gamma camera design using a metal-free 3-D position-sensitive scintillator detector. With the interspaced mosaic-patterned scintillators, one scintillator is naturally collimated by other scintillators, and the detector response is sensitive to the directional position of the incoming gamma ray, providing sufficient information for radiation source imaging in a 4π field-of-view (FOV). We have achieved fast directional gamma-ray imaging with a high-sensitivity portable collimator-less gamma-camera design for nuclear security applications . BesidesIn this work, we aim to improve the sensitivity of cardiac SPECT imaging to a new level by extending the collimator-less gamma imaging technique from 2-D directional imaging to 3-D tomographic SPECT imaging. We propose to assemble a ring-shaped system with multiple mosaic patterned, uncollimated scintillator blocks that allow a flexible system design. We perform Monte Carlo simulations to evaluate the resolution and sensitivity performance and investigate the cardiac imaging feasibility with an anthropomorphic phantom. We test the imaging feasibility with a 2-D experimental prototype.2.2.1.2.1.1.The collimator-less imaging concept is shown in 2.1.2.2.1.3.−1, which is significantly lower than that of the GAGG(Ce) scintillator (4.746 cm−1). The refraction indexes for the k-9 glass and the GAGG(Ce) scintillator are 1.5 and 1.9 respectively, which are close to each other. Such a design both allows an interspaced, mosaic-patterned spatial structure of the GAGG(Ce) scintillators and enables efficient optical-photon transportation for each scintillator to the two silicon photo-multipliers (SiPM). The entire detector block contains 38,400 spatially separated scintillator segments. In the cross-sectional plane, each scintillator bar is wrapped with total-reflective materials. The space between each scintillator bar is simply filled with air. Therefore, the electric signal readout is independently operated for each scintillator bar with two SiPMs on both ends.We propose to assemble a mosaic-patterned scintillator bar by interlacing five 1.68 mm × 1.68 mm × 2 mm GAGG(Ce) scintillator and five optical-guide segments  as shown in 2.1.4.99mTc labeled tracers. We set an intrinsic energy resolution of 20% at 140 keV for the GAGG(Ce) scintillators based on our previous experimental measurement  in the FOV to account for the photon scattering and attenuation effects during projection data acquisition. No water-filled attenuator is applied in the hot-rod and disk phantom studies. We perform multiple rounds of simulations for both hot-rod and cardiac phantoms with different activity and acquisition time combinations during the image formation process.The projection data are defined by concatenating the record counts in the detector elements of all the seven cylinder-shaped scintillator blocks into a single vector.Where 99mTc cylindrical source (180 mm (Φ) × 100 mm (L)) filling the FOV is simulated for all three phantom studies. In the cardiac phantom study, we place the water-filled cylindrical attenuator  in the FOV to account for the attenuation and scattering effect. In each study, we acquire around 2 × 1012 events so that the impact of noise is minimal. Each recorded list-mode event contains the emission and detection position information during the simulation process. The system matrix [jth voxel detected in the ith detector bin is approximated by the total number of recorded events in the ith detector bin and emitted from jth voxel. We perform long-time Monte Carlo simulations to derive system matrices for the three phantom studies . A unifo99mTc-sestamibi tracer uptake in the myocardium  algorithm .q and k are indices of subsets and iterations, respectively. where In all cases, we empirically determine an iteration number that represents an optimal trade-off between resolution and noise. The iteration numbers for the hot-rod, disk, and cardiac phantom study are 50,10, and 50, respectively. We also apply a post-reconstruction Gaussian filter with empirically chosen parameters. Specifically, the Gaussian filter with a kernel size of 7 and FWHM of 2 mm is applied in hot-rod and disk image processing. The Gaussian filter with a kernel size of 7 and FWHM of 7 mm is applied in cardiac image processing.2.2.2.2.1.We perform proof-of-concept experiments with a mosaic-patterned detector block developed in our lab . The det4 reflector . The mosaic-patterned scintillator block is optically coupled to two 16 × 16 SiPM boards  at both ends. Each SiPM board is connected to one front-end board with customized ASIC chips  algorithm, i.e., using one subset in 3.3.1.The sensitivity was determined as the number of recorded events detected from the detector bins divided by the number of emitted photons in each voxel during the Monte-Carlo simulation. 99mTc from the simulation is shown in The energy spectra of In the cardiac phantom study , the ima3.2.99mTc is shown in The experimentally measured energy spectra of 4.In this study, we propose a novel cardiac SPECT imaging system design with only scintillators and without heavy metal materials. The key innovation is the mosaic-patterned scintillator assembly that allows one scintillator to be naturally collimated by other scintillators in front of it, enabling the determination of a photon’s pathway without sacrificing other photons as a mechanically collimated SPECT usually does. Therefore, it is possible to significantly improve the imaging speed for cardiac SPECT imaging.Current work and two previous studies , 41 fromWe have investigated the feasibility of simulation and proof-of-concept experiments. In simulations, we designed a half-ring cardiac SPECT with 7 mosaic-patterned detector modules and demonstrated that the system offers satisfactory image quality at routine tracer injection dose levels clinically. The image resolution is comparable to state-of-the-art dedicated cardiac SPECT systems. The reported sensitivity in the designed system (16.31% ± 8.85% in a 180 mm (Φ) × 100 mm (L)) is extremely high. However, since the collimation geometry and detection efficiency vary from one scintillator to another in a complicated way, one cannot simply compare the absolute sensitivity to a conventional SPECT with parallel-hole or pinhole collimation. Further research is required to explicitly analyze the image signal-to-noise property of the collimator-less SPECT system. However, the cardiac phantom study in Our imaging system relies on photons interacting with the scintillator material, potentially producing scattered events across multiple scintillators via Compton scattering. However, our Monte Carlo simulations indicate that the detected Compton-scattering events in our proposed scintillator block are approximately 9.4% in an energy window between 112 keV and 168 keV. Of these events, around 3% result in inter-crystal scatter events, making the impact of scattering events insignificant in our system.This study has several limitations. Firstly, the cardiac phantom study shows that scattering events do not significantly degrade image quality in a uniform water phantom of fixed size. However, we did not consider the impact of non-uniform body attenuation and scattering in our simulations. Scattering can have a more pronounced effect on patients with a large BMI. Secondly, the residual tracer activity out of the FOV (such as liver) is not included as well, which may cause further image quality degradation. Thirdly, our currently available detector only implements a mosaic-patterned design in the trans-axial direction, which differs from the proposed imaging system design. Furthermore, the experiments we conducted were in a planar field of view. Further work is ongoing to assemble a 3-D imaging system that matches the simulation design and investigate the impact of non-uniform body attenuation and scattering in different patient sizes.5.We propose a novel collimator-less cardiac SPECT system by using a mosaic-patterned scintillator block design that allows detector collimation through other detectors in the front. We propose a half-ring cardiac SPECT design with 7 mosaic-patterned detector modules. The simulation study demonstrates that 6-mm hot rod separation and super high detection efficiency (16.31 ± 8.85%) are achievable. The prototype experiment demonstrates the feasibility of multi-point-source imaging with an 8-mm point-source separation capability. The proposed cardiac SPECT allows myocardial SPECT scan in less than a minute with a highly flexible and scalable system structure. We conclude that it is possible to achieve high-performance SPECT imaging without a heavy-metal collimator, and our work opens the way to a very fast SPECT MPI scan with reasonable resolution.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RW: methodology, validation, formal analysis, investigation, and writing – original draft. DZ: methodology and formal analysis. YH: methodology and investigation. ZL: project administration. TM: conceptualization, supervision, writing – review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.